Simian virus 40 (SV40) large T antigen (TAg), both free and bound to mature 70S and replicating 90S SV40 chromosomes, was prepared from lytically infected cells. The relative reactivity of the different TAg-containing fractions toward 10 monoclonal antibodies directed against three different regions in SV40 TAg and toward an antibody against the p53 protein was measured. The results for free TAg indicated that all of the determinants in both the amino-terminal (0.65 to 0.62 map units) and carboxy-terminal (0.28 to 0.17 map units) regions were highly reactive, whereas all five determinants located between 0.43 and 0.28 map units in the midregion of TAg were poorly reactive. For TAg bound to replicating chromosomes, all but one of the antibodies specific for TAg were highly reactive. Thus, antigenic sites in the middle of TAg, the region important for nucleotide binding and ATP hydrolysis (an activity required for viral DNA replication), were more accessible in TAg-replicating DNA complexes. As replicating molecules matured into 70S chromosomes, three or more determinants at different locations in TAg bound to chromatin became two- to fivefold less reactive, indicating other changes in TAg structure. Overall, at least nine different antigenic determinants in the TAg molecule were identified. Anti-p53 was reactive with about 10% of the free TAg and the same amount of SV40 chromosomes of all ages, suggesting that p53-TAg complexes are not preferentially associated with either replicating or mature viral chromosomes. When the reactivity of both mature and replicating labeled SV40 chromosomes with polyclonal tumor anti-T was measured as a function of time after purification, TAg bound to mature chromosomes appeared to dissociate about fourfold faster than that bound to replicating chromosomes. The relative amount of TAg in various subcellular fractions was measured by an enzyme-linked immunosorbent assay. Approximately 1.3% of the total TAg was estimated to be associated with SV40 chromosomes in infected cells. Based on the relative amounts of TAg and viral DNA in the 70S and 90S fractions, replicating chromosome-TAg complexes were estimated to bind 4.8 times more TAg per DNA molecule, on the average, than mature chromosome-TAg complexes. Together, these results are consistent with major differences in TAg structure when free and associated with replicating and nonreplicating SV40 chromosomes.